Presently, numerous test devices are available to simply and rapidly analyze body fluids for the presence or absence of a predetermined soluble constituent. For example, tests are available to detect glucose, uric acid or protein in urine, or to detect glucose, triglycerides, potassium ion or cholesterol in blood. Historically, assays of a whole blood sample for a predetermined soluble constituent are the most difficult tests to design.
The cellular components of whole blood, and especially the red blood cells, are the primary interfering substances in assays for a soluble constituent of whole blood. Most simple blood tests are chromogenic, whereby a predetermined soluble constituent of the whole blood interacts with a particular reagent either to form a uniquely-colored compound as a qualitative indication of the presence of absence of the constituent, or to form a colored compound of variable color intensity as a quantitative indication of the presence of the constituent. The deep red color of the whole blood sample substantially interferes with these chromogenic tests, and therefore the highly-colored red blood cells usually are separated from the plasma or serum before the blood sample is assayed for a predetermined soluble constituent.
The presence of red blood cells also can interfere with various nonchromogenic blood assays, whereby the assay results are either inconsistent or, if consistent, are inaccurate. Furthermore, other cellular components, including the white blood cells, also can interfere in standard chromogenic blood assays. Therefore, to achieve a reliable assay for a predetermined soluble constituent of whole blood, it is essential to separate the serum or plasma from the cellular components of whole blood prior to analyzing the whole blood sample for the predetermined soluble constituent.
Conventionally, the plasma or serum is separated from the cellular material of whole blood by centrifugation. The cellular material collects at the bottom of the centrifuge tube and the supernatant plasma or serum is decanted. Accordingly, the interfering cellular components of whole blood are sufficiently removed such that a substantial background interference is avoided. However, the centrifuge method has the major disadvantages of requiring a relatively large blood sample, usually from about 0.1 ml to about 5 ml, and a long centrifuge time of approximately 5 to 10 minutes. Furthermore, the centrifuge method requires several manipulative steps. Consequently, a laboratory technician may contact a potentially-infectious blood sample or contact laboratory equipment contaminated by the relatively large blood sample.
Overall, the centrifuge method is best suited for large, automated laboratories that assay a large number of blood samples, and for institutions, such as hospitals, that do not require assay results in a matter of minutes. Many small laboratories and private medical offices do not have a centrifuge or other blood separator on site. Therefore, simple chromogenic tests cannot be performed quickly, safely and easily on site and the whole blood sample is sent to an outside laboratory for efficient and safe separation and assay. As a result, the assay results are available in hours or days as opposed to minutes.
Accordingly, investigators have continually sought a device and method of quickly, safely and easily separating essentially all of the interfering cellular components of whole blood from the plasma or serum such that the identity and concentration of soluble constituents in the plasma or serum are not altered. Consequently, an assay for a predetermined constituent of the plasma or serum is trustworthy, accurate and free from interference by the cellular components of the whole blood. Investigators have provided several methods and devices for separating the interfering cellular components of whole blood from the plasma or serum. However, each method and device possessed at least one disadvantage that made the method or device inaccurate, cumbersome of impractical in assaying a whole blood sample for a predetermined soluble constituent.
Methods other than centrifugation have been used to separate the cellular components of a small whole blood sample from the serum or plasma. One of the simpler methods, as disclosed by Adams et al. in U.S. Pat. No. 3,092,465, used a bibulous, or moisture absorbing, matrix that is impregnated with a chromogenic testing reagent and coated with a semipermeable barrier. The semipermeable barrier screens the cellular components of the whole blood sample and permits passage of the smaller, soluble molecules and ions to contact the chromogenic testing reagent incorporated into the bibulous matrix. In the case of a positive test, the essentially colorless plasma or serum interacts with the chromogenic testing reagent to produce a color in the bibulous matrix. The color is observed by rinsing or wiping the cellular material retained on the semipermeable barrier from the test device. However, the rinsing of wiping technique is cumbersome and laborious, and assay interference is possible if the red blood cells are not completely wiped or rinsed from the semipermeable barrier. In addition, the possibility of technician contact with the potentially-infectious blood sample is high. Mast, in U.S. Pat. No. 3,298,789, discloses a similar device, wherein a film of ethylcellulose is utilized as the semipermeable barrier. Sodickson, in U.S. Pat. No. 4,059,405, discloses separation of the cellular components from the blood plasma or serum with an ultrafiltration membrane.
Fetter in U.S. Pat. Nos. 3,552,925 and 3,552,928 discloses another method and device to assay small whole blood samples for soluble constituents. Fetter describes a test device having a bibulous matrix impregnated with a nonvolatile inorganic salt or an amino acid at a first region on the matrix and impregnated with a test reagent at an adjacent second region of the matrix. A whole blood sample is introduced onto the bibulous matrix such that the whole blood first contacts the first region of the bibulous matrix including the inorganic salt or amino acid. The salt or amino acid precipitates the cellular components from the blood, and the plasma or serum then migrates to the test reagent-impregnated second region of the bibulous matrix for a chromogenic interaction with the test reagent. The salts or amino acids used in this process effectively separate the red blood cells from the whole blood sample, but also introduce contaminating ions or molecules into the plasma or serum and precipitate a portion of the soluble plasma or serum constituents. Therefore, the method of Fetter may suffer from the disadvantage that a quantitative assay for a predetermined soluble constituent of the plasma or serum is unreliable because the plasma or serum no longer includes the true concentration of the predetermined soluble constituent, or constituents, of interest.
Another prior art method of separating the cellular components of whole blood from the plasma or serum was disclosed by Vogel et al., U.S. Pat. No. 4,477,575, describing a process and a composition for separating plasma or serum from whole blood using a layer of glass fibers having a defined average diameter and density. In addition to the defined glass fiber parameters, the amount of plasma or serum that can be separated is limited to at most 50%, and preferably less than 30%, of the absorption volume of the glass fibers. Otherwise, whole blood, containing approximately 50% filterable cellular material, effectively clogs the glass fiber layer. Therefore, the method requires a high ratio of glass fibers to whole blood volume. Vogel et al. do not teach or suggest including an agglutinin, a thrombin or a thrombin-like compound, or a nonhemolytic surfactant, in the layer of glass fibers.
In other prior art methods, the whole blood is diluted before assaying for a predetermined soluble plasma or serum constituent. The dilution of whole blood is burdensome because an extra manipulative step is required, and dilution introduces the possibility of assay error because of an incorrect dilution of the blood sample. The possibility of technician contact with the potentially infectious blood sample also is increased. For example, German Patent Publication No. DE-OS 34 41 149 discloses a method of separating plasma or serum from whole blood by passing the whole blood through a lectin-impregnated matrix that is repeatedly rinsed with a diluent to dilute the plasma or serum before the assay is performed. The agglutinating ability of the lectin was enhanced by including a cationic polymer in the matrix. However, the possibility of imprecise dilution can result in an inaccurate assay for the plasma or serum constituent of interest. German Patent Publication No. DE-OS 34 41 149 neither teaches nor suggests that a nonhemolytic surfactant can enhance the ability of a lectin to agglutinate cellular material. The use of a lectin or a polymeric amino acid to separate the cellular material from a whole blood sample is also disclosed in European Patent Application No. 84307633.2.
In developing a method and device for separating and assaying small whole blood samples, a primary consideration is the degree of sophistication of the technician performing the assay. Often it is desirable to have relatively untrained personnel perform routine assays and obtain accurate quantitative results. Therefore, it is important that the assay method include a minimum of manipulative steps, be free of possible interferences or contamination, minimize or eliminate the possibility of laboratory personnel physically contacting the blood sample, and provide for easy measurement. For instance, among the several possible manipulative steps, the dilution of the whole blood, or the plasma or serum, prior to the actual assay introduces the most probable step for assay error or personal contact with the blood sample. Another common manipulative error is the incomplete wiping or rinsing of the cellular components of whole blood from the surface of a device that utilizes a cell-impermeable membrane to separate the cellular components from the plasma or serum of whole blood.
Therefore, a need exists for a method and device to efficiently separate and accurately assay small volumes of whole blood. The method preferably avoids a distinct manipulative step to separate the cellular components from the plasma or serum prior to the assay. Furthermore, in order to avoid dilution errors, the method preferably allows the assay of undiluted plasma or serum. It also is desirable to provide a blood separation and blood assay method that protects the technician from contact with the blood sample; that avoids the time delays of the present methods; and that yields accurate and reproducible results.
The ideal method includes withdrawing a whole blood sample in a "noninvasive" amount, such as a pin prick drop, and immediately depositing the undiluted whole blood sample on a test device that both separates the cellular components from the undiluted plasma or serum, and assays the undiluted plasma or serum for the presence or concentration of a predetermined soluble constituent within minutes. Alternatively, the test device can contact a finger puncture and withdraw a fresh, undiluted blood sample from the wound for analysis. Such a separation and assay method and device allow medical personnel to perform whole blood analyses on a more routine and more confident basis.
Consequently, investigators have attempted to develop test devices that include an element to separate, collect and retain the cellular components of whole blood. In addition, some test devices were developed wherein the cell-separating element then is physically disconnected from the test device and discarded before assaying the plasma or serum for a particular constituent. For example, the previously-mentioned Vogel et al. U.S. Pat. No. 4,477,575 discloses a device wherein a glass fiber cell-separating layer is disposed over a reaction layer, such that the separating layer can be removed from the reaction layer. The reaction layer then is examined for response to a particular plasma or serum constituent. However, the glass fiber separating layer of the Vogel device requires the disadvantageous high ratio of glass fiber to blood described above. Consequently, a specific volume of blood must be pipetted onto the test device, thereby adding a time-consuming manipulative step that can result in operator error and erroneous assays. Such a manipulative step also can lead to reduced operator safety because of potential physical contact between the operator and the blood sample.
Rothe et al., in U.S. Pat. No. 4,604,264, discloses a hinged assay device. The device disclosed by Rothe et al. includes a separating layer consisting of a glass fiber fleece, wherein the whole blood is applied near the end of the separating layer distant from the hinge area of the device. The separating layer includes neither an agglutinin, like a lectin, nor a coagulant, like a thrombin or a thrombin-like compound, nor a nonhemolytic surfactant. As the blood permeates through the separating layer, the cellular components are separated from the serum or plasma. The serum or plasma then migrates towards the hinge of the device to an area of the separating layer underneath a reaction/indicator layer that is disposed above the separating layer and secured to the separating layer by a hinge. By pressing down on the reaction/indicator layer, contact between the lower face of the reaction/indicator layer and the separating layer allows the reaction/indicator layer to absorb the serum or plasma for reaction with reagents in the reaction/indicator layer. Assay detection, such as a color change, is achieved by observation through a transparent film disposed on the upper face of the reaction/indicator layer.
Kennedy et al., in application PCT/US86/02192, disclose a disposable dry phase test stick having a reactive area covered by a semipermeable membrane. The semipermeable membrane separates cellular and particulate matter from whole blood and allows the plasma or serum to contact the reactive area. The semipermeable membrane is detachable from the reactive area to remove cellular components and particulate matter from the device and to expose the reactive area for examination of a response to a particular analyte. The semipermeable membrane is a hydrophobic polytetrafluoroethylene material rendered hydrophilic with a surfactant or a soap-like wetting agent. Kennedy et al. disclose that the surfactant, either a nonionic surfactant, anionic surfactant, or cationic surfactant, is included to make the semipermeable membrane hydrophilic and to distribute blood sample so that the blood sample spreads rapidly and evenly through the semipermeable membrane. However, Kennedy et al. do not teach or suggest that a surfactant improves the ability of a small amount of an agglutinin or a coagulant to separate the cellular components from a test sample. In contrast to the present invention, wherein an agglutinin or a coagulant is utilized in a low amount, and in conjunction with a nonhemolytic surfactant, to separate the cellular material, Kennedy et al. teach the separation of the cellular material by a hydrophobic semipermeable membrane, absent an agglutinin or a coagulant, made hydrophilic with a surfactant. Furthermore, the cationic and anionic wetting agents disclosed by Kennedy et al. can remove particular noncellular components, such as potassium ions, from the plasma or serum.
Another prior art patent directed to separating the cellular components of whole blood from the plasma or serum is Rapkin et al. U.S. Pat. No. 4,678,757, wherein a carbohydrate-treated permeable carrier is used to separate the cellular components of whole blood from the plasma or serum. The plasma or serum of the whole blood then contacts a reagent-treated permeable carrier for assay of a particular blood constituent. In this device, the cellular components collect and are retained at the bottom edge of the carbohydrate-tested carrier, and the plasma or serum permeates through the carbohydrate-treated carrier to contact the reagents necessary for the assay. Assay results are determined by observation through a transparent material covering the permeable layers. Rapkin et al. disclose that sugars, like mannitol, sucrose, glucose and sorbitol, are suitable carbohydrates. Rapkin et al. do not teach incorporating a nonhemolytic surfactant, or an agglutinin or a coagulant, in the carbohydrate-treated permeable carrier.
In addition, Terminiello et al., in U.S. Pat. No. 4,774,192, disclose a porous membrane having a porosity gradient such that the cellular components of whole blood are retained in an area of the membrane having a low porosity. The porous membrane is conditioned with a protein, a sugar, a polyethylene glycol, a polypyrolidone or similar compound to reduce the void space within the matrix membrane (i.e., to lower the porosity of the membrane) and to promote absorption of the fluid portion of the test sample. Therefore, the serum or plasma can flow through the area of low porosity to contact assay reagent components incorporated into an area of the membrane having a high porosity. G. Rayman and J. L. Day, in the publication "New Device to Improve the Accuracy of Bedside Blood Glucose Tests", The Lancet, Nov. 12, 1988, pp. 1107-1109, describe a disposable test strip for glucose wherein the surface of the strip is wiped to remove the cellular components of the whole blood from the strip. However, in the Terminiello et al. device, higher molecular weight soluble plasma components, such as cholesterol, may not completely permeate through the low porosity area of the membrane; and in the Rayman and Day device the cellular components may not be completely wiped from the surface of the strip. Therefore, in each device, inaccurate and unreliable assays are possible. Neither disclosure suggests that a nonhemolytic surfactant can be included in a separating layer such that the amount of agglutinin or coagulant in the separating layer can be reduced substantially. Other patents directed to assaying whole blood for a predetermined soluble constituent wherein the cellular components are separated from the plasma include: Stone, U.S. Pat. No. 3,607,093; Figueras, U.S. Pat. No. 4,144,306; and Pierce et al., U.S. Pat. No. 4,258,001.
Therefore, because of the disadvantages present in the above-cited methods and test devices, it is apparent that a simple and effective method of separating the cellular components of whole blood to provide essentially cell-free, unaltered and undiluted plasma or serum is needed. Accordingly, the method and device of the present invention allow the safe, accurate and economical assay of a whole blood sample, or other biological fluid sample, for a predetermined soluble constituent by utilizing a filter pad having incorporated therein: a) a separating reagent, such as an agglutinin, like a blood type non-specific lectin; a coagulant, like a thrombin or a thrombin-like compound; or a combination thereof; and b)a nonhemolytic surfactant, like an ethoxylated or propoxylated nonionic or anionic surfactant, to achieve essentially complete separation of the cellular components of whole blood from the plasma or serum. The filter pad is in contact with a test pad incorporating the necessary reagents to assay for the predetermined plasma or serum constituent of interest. The essentially cell-free plasma or serum migrates through the filter pad to contact the test pad. An interaction between the predetermined constituent of interest and the assay reagents produces a detectable response, such as a color transition, free from interferences attributed to highly-colored cellular components.
The method and device of the present invention allow the assay of whole blood without resorting to lengthy and expensive wet phase assays and without resorting to the extra manipulative step of diluting the test sample. The cell-free plasma or serum that saturates the test pad is unaltered and undiluted, thereby allowing a more accurate and trustworthy assay for a predetermined soluble constituent. The method and device of the present invention also eliminate the disadvantages of hematocrit sensitivity, technique sensitivity due to wiping or rinsing the cellular components from the test device, and disposal of the cellular components.
Furthermore, in accordance with one embodiment of the present invention, after the whole blood sample has saturated the filter pad and the test pad, the filter pad retaining the cellular components of the blood can be physically disconnected from the test device, and thereby exposing the test pad of the device. The test pad, saturated with undiluted and unaltered plasma or serum, then is examined for a response to a predetermined plasma or serum constituent by standard dry phase chemistry test strip procedures. Alternatively, in another embodiment, the filter pad is not removed from the test device, and a surface of the test pad free from contact with the filter pad is examined for a response, such as examination through a transparent support.
In accordance with another important feature of the present invention, the device essentially precludes contact between the technician and the blood sample. The blood sample is absorbed into the filter pad in such a manner that excess blood sample does not remain on an outside surface of the device. In addition, the technician need not wipe or rinse the cellular components from the device before examination of the device for a response. Consequently, the device essentially eliminates the possibility of contact between the technician and a potentially infectious blood sample.
As a result of the present invention, the assay of plasma or serum for a predetermined soluble constituent is accurate and reliable because the interferences attributed to the highly-colored cellular components are essentially eliminated. Prior methods and devices rely either upon wiping the cellular components from the surface of the analyte detection device or upon immobilizing the cellular components, physically or chemically, in an area of the analyte detection device distant from the actual assay area. As will be demonstrated more fully hereinafter, the device of the present invention provides an accurate and economical method of first separating the cellular components of whole blood from the plasma or serum by an improved filter pad.
Unexpectedly, it has been found that the filter pad utilized in the present invention effectively separates the cellular components from a whole blood sample by incorporating a relatively small amount of an agglutinin, like a lectin; a coagulant like a thrombin or a thrombin-like compound, or a combination thereof, in the filter pad. Surprisingly, only a relatively small amount of agglutinin or coagulant is incorporated into the filter pad because a nonhemolytic surfactant also is incorporated into the filter pad. Furthermore, the serum or plasma is distributed evenly throughout the entire test pad, therefore the assay response is homogeneous throughout the test area of the device.
U.S. patent application Ser. No. 063,680, filing date Jun. 19, 1987, and commonly assigned to the assignee of the present invention, discloses that an agglutinin or a coagulant included in a filter pad effectively separates the interfering cellular material from a whole blood sample. However, the filter pad was impregnated with a relatively large amount of expensive agglutinin or coagulant to achieve a satisfactory separation. In addition, because the purity or activity of an agglutinin or coagulant can vary, including a large amount of agglutinin or coagulant in a filter pad can lead to clogging of the filter resulting in inconsistent or irreproducible results. Therefore, in accordance with an important feature of the present invention, it has been found that incorporating a nonhemolytic surfactant into the filter pad allows a significant reduction in the amount of agglutinin or coagulant incorporated in the filter pad. Consequently, the filter pad and test device of the present invention are more economical, separate essentially all of the cellular components from a whole blood sample, and provide more consistent and reproducible assays.